Protein sequence of the plant toxin gelonin

ABSTRACT

This invention relates to substantially purified gelonin, toxic fragments thereof, the DNA sequences encoding gelonin and use of the DNA for producing, by recombinant technology, gelonin, toxic fragments thereof and fusion proteins. More specifically, the invention relates to the primary amino acid sequence of gelonin, and of the DNA encoding said gelonin and the production of synthetic gelonin and toxic fragments thereof.

This is a reissue of Ser No. 08/254,662 filed Jun. 6, 1994, now U.S.Pat. No. 5,631,348 which is a continuation of application Ser. No.08/119,899 filed on Sep. 10, 1993, now abandoned, which is acontinuation of U.S. Ser. No. 07/908,959 filed Jul. 6, 1992, abandoned,which is a continuation of U.S. Ser. No. 07/567,220, filed Aug. 14,1990, now abandoned.

TECHNICAL FIELD

This invention relates to substantially purified gelonin, toxicfragments thereof, the DNA sequences encoding gelonin and use of the DNAfor producing, by recombinant technology, gelonin, toxic fragmentsthereof and fusion proteins. More specifically, the invention relates tothe primary amino acid sequence of gelonin, and of the DNA encoding saidgelonin and the production of synthetic gelonin and toxic fragmentsthereof.

BACKGROUND ART

A major challenge for the design of a drug for treatment of any diseaseis specificity and efficacy. Various drugs available for the treatmentof cancer suffer from problems of this nature. The concept of targetingtoxic drugs selectively to certain tumors has been a subject of intenseresearch in the last few years (Thorpe (1985) Biol Clin Applications84:475-512; Moller ed. (1982) Immun. Rev. 62:1-215). Recently bothmonoclonal and polyclonal antibodies, lectins, lymphokines and hormoneswhich recognize specific determinants on the surface of the tumor cellhave been used as carriers to deliver toxic agents into the cell, wherethe latter can exert their cytotoxic potential (Blattler, et al. (1985)Biochemistry 24:1517-1524; Frankel, et al. (1985) J. Biol. Res. Modif.4:437-446; Reimann, et al. (1988) J. Clin. Invest. 82:129-138; Schwartzand Vale (1988) Endocrinology 122:1695-1700; Scott, et al. (1987) JNatl. Cancer Inst. 79:1163-1172; Singh, et al. (1989) Biol. Chem.264:3089-3095; Srinivasan, et al. (1985) FEBS Letters 192:113; Schwartz,et al. (1987) Endocrinology 121:1454-1460). Toxic moieties thus farinvestigated with these delivery agents include radionuclides (Ghose, etal. (1967) Br. Med. J. 1:90-96), cytotoxic drugs commonly employed incancer chemotherapy (Thorp and Ross (1982) Immun. Rev, 62:119-157;Deweger, et al. (1982) Immun. Rev. 62:29-45; Arnon and Sela (1982)Immun. Rev. 62:5-27; Pimm, et al. (1982) Cancer Immun. Immunotherap,12:125-134; Rowland and Axton (1985) Cancer Immun. Immunotherap. 19:1-7)and proteins derived from bacteria and plants such as diptheria or ricin(Jansen, et al. (1982) Immun. Rev. 62:185-216; Raso (1982) Immun. Rev.62:93-117; Vitetta, et al. (1982) Immun. Rev. 62:159-183; Nelville andYoule (1982) Immun. Rev. 62:47-73; Thorpe, et al. (1981) Eur. J.Biochem. 116:447-454). A specific molecule is designed by replacing thenonspecific B chain with an antibody or a hormone.

Bacterial and plant toxins, such as diphtheria toxin (DT), Pseudomonasaeruginosa toxin A, abrin, ricin, mistletoe, modeccin, and Shigellatoxin, are potent cytocidal agents due to their ability to disrupt acritical cellular function. For instance, DT and ricin inhibit cellularprotein synthesis by inactivation of elongation factor-2 andinactivation of ribosomal 60s subunits, respectively (Bacterial Toxinsand Cell Membranes, Eds. Jelajaszewicz and Wadstrom (1978) AcademicPress, p. 291). These toxins are extremely potent because they areenzymes and act catalytically rather than stoichiometrically. Themolecules of these toxins are composed of an enzymatically activepolypeptide chain or fragment, commonly called “A” chain or fragment,linked to one or more polypeptide chains or fragments, commonly called“B” chains or fragments, that bind the molecule to the cell surface andenable the A chain to reach its site of action, e.g., the cytosol, andcarry out its disruptive function. The act of gaining access to thecytosol is called variously “internalization”, “intoxication”, or“translocation”. These protein toxins belong to a class bearing twochains referred to as A and B chains. The B chain has the ability tobind to almost all cells whereas the cytotoxic activity is exhibited bythe A chain. It is believed that the A chain must be timely liberatedfrom the B chain-frequently by reduction of a disulfide bond-in order tomake the A chain functional. These natural toxins are generally notselective for a given cell or tissue type because their B chainsrecognize and bind to receptors that are present on a variety of cells.

The availability of a toxin molecule which is not cytotoxic to a varietyof cells when administered alone has been limited. Utilizing certainnaturally occurring single chain toxin molecules which do not themselvesbind to cell surface receptors and, therefore, are not normallyinternalized by cells, has provided toxic molecules which are relativelynon-toxic to most, if not all, cells when administered alone. Suchnaturally occurring single chain toxins known to date, include, but arenot limited to, pokeweed antiviral protein (Ramakrishnan and Houston(1984) Cancer Res. 44:201-208), saponin (Thorpe, et al. (1985) J. Natl.Cancer Inst. 75:151-159), and gelonin (Stirpe, et al (1980) J. Biol.Chem. 255:6947-6953). These proteins are nontoxic to cells in the freeform, but can inhibit protein synthesis once they gain entry into thecell. However, the availability of these single chain toxins insubstantially pure form is limited due to the fact that they must bepurified from plant sources in which they occur in relatively lowamounts and the reproducibility of the concentration of the toxin in theplants is dependent upon plant growth conditions and plant harvestconditions.

Gelonin is a single chain polypeptide isolated from seeds of a plant,Gelonium multiforum, having a molecular weight of approximately28,000-30,000 kd. Gelonin is a basic glycoprotein with an approximateisoelectric point of 8.15 and contains mannose and glucosamine residues(Falasca, et al. (1982) Biochem J, 207:505-509). In contrast to otherplant and bacterial toxins, this protein is not toxic to cells byitself, but when delivered to cells through a carrier, it damages the60s ribosomal subunit. In vivo and in vitro biological data suggest thatgelonin is equivalent or superior to other plant toxins. In fact, theresults of a comparison of gelonin conjugates in vitro and in vivo withother A chain conjugates indicated that gelonin had similar potency,better selectivity, better tumor localization, and more significanttherapeutic effects (Sivan, et al (1987) Cancer Res. 47:3169-3173).However, the availability of a reproducible, readily accessible supplyof gelonin from natural sources is limited. In addition, thepurification of gelonin from plant sources is difficult and the yield isvery low.

Gelonin by itself has been shown to be abortifacient in mice andenhances antibody dependent cell cytotoxicity (Yeung, et al (1988)Internatl. J. Peptide Protein Res. 31:265-268).

Several investigators have utilized gelonin as a cytotoxic agentchemically attached to monoclonal antibodies or to peptide hormonecellular targeting ligands. However, chemical modification of geloninand cellular targeting moieties can reduce targeting efficiency andcytotoxic potential of gelonin itself. Furthermore, natural sources ofgelonin are subject to variability in harvesting and plant growth whichcan affect gelonin cytotoxic activity. The ability to produce asynthetic gelonin toxin, chemically or utilizing recombinant technology,provides a plentiful, reproducible source of the toxin.

SUMMARY OF THE INVENTION

The present invention provides substantially pure gelonin having theamino acid sequence shown in FIG. 1. The present invention also providesthe DNA sequence for gelonin shown in FIG. 2. Utilization of thesequences of the present invention to produce substantially pure geloninin plentiful amounts by recombinant technology provides abundant amountsof the toxin which were not heretofore available from natural sources.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of gelonin.

FIG. 2 demonstrates the cDNA encoding for gelonin.

FIG. 3 demonstrates the homology of the gelonin amino acid sequence withthe sequence of trichosanthin, Ricin A chain, Agglutinin precursorisolated from Castor bean and Abrin A chain.

FIG. 4 demonstrates the HPLC profile of CNBr fragments.

FIGS. 5A, 5B and 5C demonstrate the HPLC profile of (A) Lys-c, (B)Staphylococcus protease, and (C) Hydroxylamine digests of gelonin.

FIGS. 6A, 6B, 6C and 6D , 6D and 6E demonstrate the hydrophobicity plotsof gelonin (A), trichosanthin (B), abrin (C), ricin (D), and agglutininprecursor (E).

DETAILED DESCRIPTION OF THE INVENTION

The term “substantially pure” when applied to the gelonin protein of thepresent invention means that the polypeptide is essentially free ofother plant proteins normally associated with the gelonin in its naturalstate and exhibiting reproducible electrophoretic or chromatographicresponse, elution profiles, and toxic activity. The term “substantiallypure” is not meant to exclude artificial or synthetic mixtures of thegelonin protein with other compounds.

Gelonin was purified from the seeds of the plant Gelonium multiforum bytechniques known to those of skill in the art. The amino acid sequencewas determined utilizing a modification of the Edman degradation method.

Samples of gelonin were applied to the reverse phase reaction chamberand subjected to Edman degradation. The N-terminal of gelonin was foundto be heterogeneous (½ of the molecules of the protein were apparentlyone amino acid shorter than the others). This heterogeneity made itdifficult to sequence much more than 40 cycles. Therefore, in order todetermine further amino acids in the sequence, enzymatic cleavage wasperformed.

Internal sequence of proteins is generally obtained by digesting orcutting up the large protein molecule into smaller pieces with acombination of enzymes and chemical cleavages. When native gelonin wasexposed to various protolytic enzyzme digestions, it was found to beincompletely cleaved. This was found to be partly due to a disulfidebond in the N-terminal part of the molecule. Breaking of this bond byreduction and alkylation with iodoacetic acid yielded a fragment thatwas less soluble than the native material at the pH required forenzymatic digestion. A combination of digestion of native gelonin withtrypsin, Lysine aminopeptidase (Lysc), staphylococcal protease (V8), andchymotrypsin yielded peptides mostly from the C-terminal portion of themolecule. This indicated that the N-terminal part of the molecule (fromthe N-terminal analysis to the Asp-Ala-Pro at residue 70) was notreadily accessible by enzyme digestions.

Gelonin was cleaved with cyanogen bromide into 3 large peptides. Proteinaliquots (0.2 mg/ml) were dissolved in 70% formic acid. A crystal ofcyanogen bromide was added to the solution and the reaction allowed toproceed for at least 18 hours. The solution was then diluted with waterand was applied to a small sequencing column. After sample application,a gradient of 1% to 10% n-propanol with 0.1% TFA was used to elute theprotein fragments. The elution profile is shown on FIG. 4.

Enzymatic digestion of the whole protein or of CNBr fragments yeildedoverlapping peptides. Enzymatic digestions with Lysyl endopeptidase in0.1% SDS 100 mM Tris pH 8.0, Staphylococcus aureus protease in 0.1% SDSor trypsin in 0.1% Tween 20 were carried out. Gelonin contains onecysteine residue at position 49. Reduction and carboxymethylation yieldsa protein which recovers better on reverse phase HPLC and is moresusceptible to enzymatic digestion. Therefore, most of the enzymaticdigestions were carried out in 0.1% SDS or 0.1% Tween.

After the C-terminal 160 residues were aligned by a combination of CNBrdigests and enzymatic cleavages, the remaining unknown sequence betweenresidues 40 to 70 was determined by a combination of chemicalmodification of cysteine with iodoacetic acid and solubization of thealkylated protein with SDS. The RCM alkylated gelonin was then cleavedwith excess Lysc enzyme at 37° C. for short periods of time (1-5 hr.).The HPLC elution profile is shown on FIG. 5A.

This method yielded a new sequence that had not been seen before. Thisnew sequence showed the existence of an Asn-Gly combination. Thiscombination of amino acids is cleaveable by a chemical method usinghydroxylamine.

Hydroxylamine cleavage was carried out by adding 100 ug of gelonin tofreshly prepared hydroxylamine (2M) in 0.2 M Tris (pH 9.0) with 2M NaCl,1 mm EDTA and 10% ethanol. After incubation for 7 hours at roomtemperature, the entire reaction mixture was applied to a sequencingcolumn. The column was then washed with 1% TFA in water and eithereluted with an acetonitrile gradient or was sequenced directly as amixture. This chemical cleavage produced a large hydrophobic peptidethat contained about a 200 amino acid sequence which connected with theAsp-Ala-Pro at residue 70. The elution profile is shown on FIG. 5C.

The remaining, short section of overlapping sequence from betweenresidues 40 to 50 was determined by digesting gelonin without alkylationby Lysc in SDS. This digested away most of the C-terminal part of thematerial. Then this mixture was digested again by chymotrypsin. Theproducts of this digestion were then separated by HPLC. Sequenceanalysis of a large peptide revealed a sequence (SerThrLys) startingabout 5 amino acids in from the N terminal end of the molecule. This wasuseful in that it removed the heterogeneous part of the molecule andallowed for a longer sequence run.

Gelonin protein comprises 258 amino acids, the sequence of which isdemonstrated on FIG. 1. The amino acid sequence of gelonin was comparedto other known sequences available in sequence data banks (Genbank, PIR,EMBL) to determine whether gelonin has any areas of homology with otherproteins. Comparison of the gelonin amino acid sequence with otherproteins having known amino acid sequences demonstrated that the geloninsequence is unique. Homology of certain portions of the gelonin sequenceto portions of other proteins was detected. For instance, gelonindemonstrates a 36.0% homology with alphatrichosanthin from Trichosanthinkirilowi, 33.8% homology with Abrin A chain from Indian Liquorice, 35.2%homology with agglutinin precursor from Castor bean, 33.7% homology withRicin D, A chain from Castor bean and 27.3% homology with antiviralprotein (MAP) from Mirabills jalapa. A summary of the degree of homologyto these and other proteins is shown on FIG. 3.

Hydrophobicity plots shown on FIGS. 6A-6E demonstrate a similarity tohydrophobic regions of trichosanthin, Ricin and to other ribosomalinhibiting proteins.

A plot of the hydropathy of the gelonin structure shows a hydrophobicregion in residues 35-80 and 150-180. These are areas in whichsubstantial folding of the molecule probably occurs. This similarhydrophobic pattern is also observed for other toxins (see FIGS. 6A-6E)and may suggest that the active enzymatic center may be contained withinthese folded regions. Therefore, the active enzymatic site may not befound in a linear region of the molecule and these structures may needto be adequately folded to attain the proper enzymatic center.

Utilizing the cDNA of gelonin, recombinant gelonin can be produced.Mutations can be specifically introduced into the molecule in order toprovide recombinant gelonin lacking carbohydrate groups which canmisdirect gelonin-antibody conjugates. Recombinant gelonin molecules canbe produced by site directed mutagenesis to have greater toxic activitythan the native molecule, to be more effectively internalized once boundto the cell surface by a carrier such as a monoclonal antibody or atargeting ligand such as IL-2, EGF, IFN, etc., to resist lysosomaldegradation and thus be more stable and longer acting as a toxic moiety.

Recombinant gelonin molecules can also be engineered as fusion productsto contain other functional modalities to kill cells such as anenzymatic activity, TNF, IFN activity, a second toxic activity, such asdiptheria toxin action (wherein said second activity was through adifferent biological pathway than gelonin), thus creating a “supertoxin”or a toxin with multifunctional actions.

Fusion proteins can be engineered with gelonin to carry drugs such aschemotherapeutic agents or isotopes for radioimaging or radiotherapy.Gelonin peptides may have application as abortofacient agents, immunosuppressive agents, anticancer agents and as antiviral agents (such asan anti-HIV agent).

The following examples provide a detailed description of thepreparation, characterization, and amino acid sequence of gelonin. Theexperimental methods utilized are described in detail in the examplesbelow. These examples are not intended to limit the invention in anymanner.

EXAMPLE 1 Purification and Characterization of Gelonin

Gelonin was isolated from the seeds of the plant Gelonium multiforumessentially according to the procedure as described (Stirpe, et al.(1980) J. Biol. Chem 255 6947-6953). Briefly, gelonin was extracted fromthe seeds by homogenization in buffered saline solution (pH 7.4). Thesupernatant was concentrated after dialysis against 5 mM sodiumphosphate (pH 6.5) and the gelonin further purified by ion exchangechromatography as described below. The purity of the gelonin toxin wasassessed by high pressure liquid chromatography (HPLC) and sodiumdodecylsulphate-polyacylamide gel electrophoreseis (SDS-Page). Gelonintoxin migrated as a single band with an approximate molecular weight of29-30,000 daltons.

Gelonin toxin activity was measured as described in Example 2 by proteinsynthesis inhibition in a cell-free system.

Seeds of Gelonium multiforum were shelled and the nuts ground in ahomogenizer with eight volumes of 0.14 M NaCl containing 5 mM sodiumphosphate (pH 7.4). The homogenate was left overnight at 4° C. withcontinuous stirring, cooled on ice and centrifuged at 35,000 times g for20 minutes at 0° C. The supernatant was removed, dialyzed against 5 mMsodium phosphate (pH 6.5) and concentrated using a pm10 filter. Thesample was layered on a CM-52 ion-exchange column (20×1.5 cm)equilibrated with 5 mM sodium phosphate (pH 6.5). Material which boundto the ion exchange resin was eluted with 400 ml of 0 to 0.3 M linearNaCl gradient at a rate of 25 ml hour at 4° C. Five ml fractions werecollected. The fractions were monitored at 280 nm in aspectrophotometer. The gelonin eluted in about fractions 55-70 and wasthe last major elution peak. These fractions were pooled, dialyzedagainst 0.1 M NaCl in 0.1 M Na₂HPO₄ buffer (pH 7.4). The sample was thenapplied to a Cibacron blue sepharose column (24×2 cm) previouslyequilibrated with 0.1 M Na₂HPO₄/0.1 M NaCl buffer. The column was washedwith 3 column volumes of buffer and eluted with a 400 ml linear saltgradient (from 0.1 M NaCl to 2 M NaCl). Elution of the bound materialwas monitored by Lowry assay of the column fractions. The fractionscontaining the single protein peak were pooled and dialyzed overnight at4° C. against PBS. Gelonin toxin was purified to greater than 97% purityas estimated from silver stained PAGE. The purity and the molecularweight of each preparation was checked on high pressure liquidchromatography using a TSK 3000 gel permeation column with 50 mM sodiumphosphate buffer, pH 7.4 and 15% sodium dodecylsulphate-polyacrylamidegel electrophoresis (SDS-page). Gelonin migrated as a single band withan approximate molecular weight of 29-30,000 daltons.

EXAMPLE 2 Assay of Gelonin Activity

The gelonin activity was monitored in a cell-free protein synthesisinhibition assay. The cell-free protein synthesis inhibition assay wasperformed by sequentially adding to 50 ul rabbit reticulocyte lysate,thawed immediately before use, mixing after each addition, the followingcomponents: 0.5 ml of 0.2 M Tris HCl (pH 7.8), 8.9 ml of ethyleneglycol, and 0.25 ml of 1 M HCl).

Twenty microliters of a salt-amino acid-energy mixture (SAEM) consistingof: 0.375 M KCl, 10 mM Mg(CH₃CO₂)₂, 15 mM glucose, 0.25-10 mM aminoacids (excluding leucine), 5 mM ATP, 1 mM GTP, 50 mM Tris-HCl (pH 7.6),10 ul Creatinine phosphate-creatinine phosphokinase, 8 ul [¹⁴C] leucine(Amersham, 348 mCi/mmol), and adding 1.5 ul of solutions containingvarying concentrations of the gelonin mixture. The mixture was incubatedfor 60 minutes at 30° C. ¹⁴C-leucine incorporation was monitored in analiquot of the mixture by precipitating synthesized protein on glassfiber filters, washing in 10% TCA and acetone, and monitoring theradioactivity in a Beta-counter using Aquasol scintillation fluid.Utilizing this assay, purified gelonin had a specific activity of 4×10⁹U/mg protein. A unit of gelonin activity is the amount of geloninprotein which causes 50% inhibition of incorporation of [¹⁴C] leucineinto protein in the cell free assay.

EXAMPLE 3 Determination of Gelonin Amino Acid Sequence

The gelonin amino acid sequence was determined by the Edman degradationmethod using an automated amino acid sequencer as described in EuropeanPatent Application No. EP-257735. Large peptides and unfragmentedprotein were applied to the reverse phase portion of the sequencereaction chamber. Unwanted buffer components were washed off with excesswater. The protein or peptide sample was then sequenced by Edmanchemistry and the extracted ATZ amino acid derivatives were converted tothe PTH form by 25% TFA in H₂O at 65° C. PTH samples were identified byreverse phase analytical separation on a Np 1090 column.

In order to obtain further amino acid sequence, the protein was digestedwith various proteolytic and chemical agents and then the peptides werepurified by high performances liquid chromatography. Gelonin was foundquite resistant to the exposure of trypsin (cleaves after arginine andlysine residues) and acetyl trypsin (cleaves only after lysine residue).The protein was found resistant to as much as 5% (w/w) of the enzyme.The resistance of gelonin to the proteolytic enzyme trypsin is not dueto a lack of trypsin cleavage sites, since gelonin contains 21 lysineand 12 arginine residues. These results indicate that gelonin is perhapsa rigidly packed molecule which makes it inaccessible to proteolyticenzymes.

Since gelonin was found resistant to cleavage by proteolytic enzymes,chemical cleavage of the protein was examined.

EXAMPLE 4 CNBr Cleavage of Gelonin

Gelonin prepared as in Example 1 was dissolved in 70% formic acid. Acrystal of cyanogen bromide was added to the solution. After at least 18hours the solution was applied to either a small column (0.15 cm×5 cm)reverse phase (J. T. Baker, 15 cm C-1B bonded phase Cat II 7191-02) oranalytical (4.6×100 mm) reverse phase column. A gradient elution of 1 to70% n propanol with 1% TFA in water produced 5 peaks as shown on FIG. 6.Each of the peaks were sequenced and also used for further digestion byenzymes to piece together the entire sequence. Peak 1 was sequenceddirectly and gave a sequence starting with a Phe (F) that ran for 38residues and ending with a Glu (E). This sequence was confirmed by massspectroscopy and Lysc digestions of this isolated peptide. Peak 2 wassequenced directly and gave a sequence starting with a Val (V) that ranfor 47 cy and was not interruptible after the Ala at cy 47. Peak 3 wassequenced and gave the same sequence as peak 2. SDS gels of peaks 2 and3 as well as Lysc digestion of peaks 2 and 3 showed that peak 3contained the C-terminal CNBr peptide as well. Subsequent trypsindigestion of gelonin produced a peptide that connected these two CNBrpeptide sequences. This trypsin peptide when sequenced gave the sequenceTSGANGMFSEAVELER. Peak 4 and 5 both gave the N-terminal sequence GLDT .. . . This was used for some digestion by Lysc, ⅛, to give peptides fromits C-terminal end.

EXAMPLE 5 Enzymatic Digestion of CNBr Cleaved Gelonin

Samples of whole protein or CNBr fragments were digested with Lysylendopeptidase (Wako Chemical Dallas, Tex.) in 0.1% SDS 100 mM Tris pH8.0 or Staphylococcus aureus protease (Pierce) in 0.1% SDS or Trypsin(Sigma) in 0.1% Tween 20. Digestion mixtures were separated by HPLC andcollected peptides were sequenced on the prototype sequence usinggas-phase Edman sequencing methods.

EXAMPLE 6 Amino Acid Sequence of Gelonin

A total of 258 amino acid residue sequences were obtained followinganalysis of the CNBr fragments obtained in Example 3. FIG. 1 shows theamino acid sequence of gelonin. Gelonin contains a total ofapproximately 258 amino acid residues. The DNA sequence was deduced fromthis amino acid sequence. The degenerate DNA sequence is shown on FIG.2. Those skilled in the art will recognize that fragments andderivatives of either the gelonin amino acid sequence or the DNAsequence coding for gelonin may inhibit cellular protein synthesis butnot bind to a cell surface receptor.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth below.

What is claimed as new and is desired to be covered under Letters Patentis:
 1. Substantially pure gelonin toxin having the amino acid sequence:GlyLeuAspThrValSerPheSerThrLys 10 GlyAlaThrTyrIleThrTyrValAsnPhe 20LeuAsnGluLeuArgValLysLeuLysPro 30 GluGlyAsnSerHisGlyIleProLeuLeu 40ArgLysGlyAspAspProGlyLysCysPhe 50 ValLeuValAlaLeuSerAsnAspAsnGly 60GlnLeuAlaGluIleAlaIleAspValThr 70 SerValTyrValValGlyTyrGlnValArg 80AsnArgSerTyrPhePheLysAspAlaPro 90 AspAlaAlaTyrGluGlyLeuPheLysAsn 100ThrIleLysAsnProLeuLeuPheGlyGly 110 LysThrArgLeuHisPheGlyGlySerTyr 120ProSerLeuGluGlyGluLysAlaTyrArg 130 GluThrThrAspLeuGlyIleGluProLeu 140ArgIleGlyIleLysLysLeuAspGluAsn 150 AlaIleAspAsnTyrLysProThrGluIle 160AlaSerSerLeuLeuValValIleGlnMet 170 ValSerGluAlaAlaArgPheThrPheIle 180GluAsnGlnIleArgAsnAsnPheGlnGln 190 ArgIleArgProAlaAsnAsnThrIleSer 200LeuGlnAsnLysTrpGlyLysLeuSerPhe 210 GlnIleArgThrSerGlyAlaAsnGlyMet 220PheSerGluAlaValGluLeuGluArgAla 230 AsnGlyLysLysTyrTyrValThrAlaVal 240AspGlnValLysProLysIleAlaLeuLeu 250 LysPheValAspLysAspProGlu 260

or a fragment or derivative thereof, said fragment or derivative havingan activity which inhibits cellular protein synthesis but does not bindto a cell surface receptor.
 2. A DNA sequence of the formula: GGNYTNGAYACNGTNWSNTT YWSNACNAAR GGNGCNACNT AYATHACNTA YGTNAAYTTY 60 YTNAAYGARYTNMGNGTNAA RYTNAARCCN GARGGNAAYW SNCAYGGNAT HCCNYTNYTN 120 MGNAARGGNGAYGAYCCNGG NAARTGYTTY GTNYTNGTNG CNYTNWSNAA YGAYAAYGGN 180 CARYTNGCNGARATHGCNAT HGAYGTNACN WSNGTNTAYG TNGTNGGNTA YCARGTNMGN 240 AAYMGNWSNTAYTTYTTYAA RGAYGCNCCN GAYGCNGCNT AYGARGGNYT NTTYAATAAY 300 ACNATHAARAAYCCNYTNYT NTTYGGNGGN AARACNMGNY TNCAYTTYGG NGGNWSNTAY 360 CCNWSNYTNGARGGNGARAA RGCNTAYMGN GARACNACNG AYYTNGGNAT HGARCCNYTN 420 MGNATHGGNATHAATAARYT NGAYGARAAY GCNATHGAYA AYTAYAARCC NACNGARATH 480 GCNWSNWSNYTNYTNGTNGT NATHCARATG GTNWSNGARG CNGCNMGNTT YACNTTYATH 540 GARAAYCARATHMGNAAYAA YTTYCARCAR MGNATHMGNC CNGCNAAYAA YACNATHWSN 600 YTNGARAAYAARTGGGGNAA RYTNWSNTTY CARATHMGNA CNWSNGGNGC NAAYGGNATG 660 TTYWSNGARGCNGTNGARYT NGARMGNGCN AAYGGNAARA ARTAYTAYGT NACNGCNGTN 720 GAYCARGTNAARCCNAARAT HGCNYTNYTN AARTTYGTNG AYAARGAYCC NGAR 774 wherein R = A or GK = G or T N = any Y = C or T M = A or C S = C or G B = C, G, or T V =A, C, or G W = A or T D = A, G, or T H = A, C, or T X = unknown

or a fragment or derivative thereof, said fragment or derivative codingfor gelonin or for a polypeptide having an activity which inhibitscellular protein synthesis but does not bind to a cell surface receptor.3. The toxin of claim 1 further defined as having the amino acidsequence: GlyLeuAspThrValSerPheSerThrLys GlyAlaThrTyrIleThrTyrValAsnPheLeuAsnGluLeuArgValLysLeuLysPro GluGlyAsnSerHisGlyIleProLeuLeuArgLysGlyAspAspProGlyLysCysPhe ValLeuValAlaLeuSerAsnAspAsnGlyGlnLeuAlaGluIleAlaIleAspValThr SerValTyrValValGlyTyrGlnValArgAsnArgSerTyrPhePheLysAspAlaPro AspAlaAlaTyrGluGlyLeuPheLysAsnThrIleLysAsnProLeuLeuPheGlyGly LysThrArgLeuHisPheGlyGlySerTyrProSerLeuGluGlyGluLysAlaTyrArg GluThrThrAspLeuGlyIleGluProLeuArgIleGlyIleLysLysLeuAspGluAsn AlaIleAspAsnTyrLysProThrGluIleAlaSerSerLeuLeuValValIleGlnMet ValSerGluAlaAlaArgPheThrPheIleGluAsnGlnIleArgAsnAsnPheGlnGln ArgIleArgProAlaAsnAsnThrIleSerLeuGluAsnLysTrpGlyLysLeuSerPhe GlnIleArgThrSerGlyAlaAsnGlyMetPheSerGluAlaValGluLeuGluArgAla AsnGlyLysLysTyrTyrValThrAlaValAspGlnValLysProLysIleAlaLeuLeu LysPheValAspLysAspProGlu.


4. The DNA sequence of claim 2, further defined as having the nucleotidesequence: GGNYTNGAYA CNGTNWSNTT YWSNACNAAR GGNGCNACNT AYATHACNTAYGTNAAYTTY 60 YTNAAYGARY TNMGNGTNAA RYTNAARCCN GARGGNAAYW SNCAYGGNATHCCNYTNYTN 120 MGNAARGGNG AYGAYCCNGG NAARTGYTTY GTNYTNGTNG CNYTNWSNAAYGAYAAYGGN 180 CARYTNGCNG ARATHGCNAT HGAYGTNACN WSNGTNTAYG TNGTNGGNTAYCARGTNMGN 240 AAYMGNWSNT AYTTYTTYAA RGAYGCNCCN GAYGCNGCNT AYGARGGNYTNTTYAATAAY 300 ACNATHAARA AYCCNYTNYT NTTYGGNGGN AARACNMGNY TNCAYTTYGGNGGNWANTAY 360 CCNWSNYTNG ARGGNGARAA RGCNTAYMGN GARACNACNG AYYTNGGNATHGARCCNYTN 420 MGNATHGGNA THAARAARYT NGAYGARAAY GCNATHGAYA AYTAYAARCCNACNGARATH 480 GCNWSNWSNY TNYTNGTNGT NATHCARATG GTNWSNGARG CNGCNMGNTTYACNTTYATH 540 GARAAYCARA THMGNAAYAA YTTYCARCAR MGNATHMGNC CNGCNAAYAAYACNATHWSN 600 YTNGARAAYA ARTGGGGNAA RYTNWSNTTY CARATHMGNA CNWSNGGNGCNAAYGGNATG 660 TTYWSNGARG CNGTNGARYT NGARMGNGCN AAYGGNAARA ARTAYTAYGTNACNGCNGTN 720 GAYCARGTNA ARCCNAARAT HGCNYTNYTN AARTTYGTNG AYAARGAYCCNGAR 774 wherein R = or G K = G or T N = any Y = C or T M = A or C S = Cor G B = C, G, or T V = A, C, or G W = A or T D = A, G, or T H = A, C,or T X = unknown.


5. The gelonin toxin of claim 1 or claim 3, further defined as a fusionprotein that includes a functional modality in addition to said toxin.6. The gelonin toxin of claim 5 wherein the fusion protein is preparedby conjugation of the functional modality to the toxin.
 7. The gelonintoxin of claim 5, wherein the fusion protein is prepared by recombinanttechnology.
 8. The gelonin toxin of claim 5, wherein said functionalmodality is an antibody.
 9. The gelonin toxin of claim 5, wherein thefunctional modality is a targeting ligand.
 10. The gelonin toxin ofclaim 9, wherein the targeting ligand is IL-
 2. 11. The gelonin toxin ofclaim 9, wherein the targeting ligand is EGF.
 12. The gelonin toxin ofclaim 9, wherein the targeting ligand is IFN.
 13. The gelonin toxin ofclaim 5, wherein the functional modality is a second toxic activity. 14.The gelonin toxin of claim 13, wherein said second toxic activity isdiptheria toxin action.
 15. The gelonin toxin of claim 5 wherein thefunctional modality is TNF.
 16. The gelonin toxin of claim 5, whereinthe functional modality is a chemotherapeutic agent.
 17. The gelonintoxin of claim 5, wherein the functional modality is a radioisotope. 18.The gelonin toxin of claim 1 or claim 3, further defined as lackingcarbohydrate groups.